ELECTRON TOMOGRAPHY OF VERTEBRATE MITOTIC SPINDLES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 快速冷冻、冷冻替代固定样本的电子断层扫描将用于检查U2OS细胞中期纺锤体体积的很大一部分，随后将对在纺锤体形成过程中冷冻和固定的细胞进行类似的工作。我们的结构研究将采用改进的样品制备方法(T R&D 3A)，以及大规模蒙太奇和多个连续切片，组装成3D重建(T R&D 5D)。纺锤形微管(MTS)的轨迹最初将通过手工建模来确定，但在随后的工作中，我们希望有自动分割的软件。所得到的MT轨迹模型将用聚类分析和几个特定的应用软件工具进行研究，以将MT的组织与四种不同和流行的纺锤体形成模型的预测进行比较：经典的中心体起始模型，MT相关的电机酶导致纺锤体MT自组织的观点，MT起始和/或稳定响应于来自染色体的梯度的观点，以及非/MT“矩阵”指导MT的形成和组织的概念。这些数据应该会消除或至少侵蚀一个或多个这样的模型，并肯定会为解释我们的实验室和其他地方正在进行的关于纺锤体形成和功能的功能实验提供基线结构信息。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Electron tomography of rapidly frozen, freeze-substitution fixed samples will be used to examine a significant fraction of the volume of metaphase spindles from U2OS cells, followed by comparable work on cells frozen and fixed during spindle formation. Our structural studies will employ improved methods for sample preparation (TR&D 3A) and both large-scale montages and multiple serial sections, assembled into 3D reconstructions (TR&D 5D). The trajectories of spindle microtubules (MTs) will initially be determined by hand modeling, though in subsequent work we hope to have software for automatic segmentation. The resulting models of MT trajectories will be studied with cluster analysis and several application-specific software tools to compare MT organization with the predictions from four distinct and popular models for spindle formation: the classic, centrosome-initiation model, the idea that spindle MTs self-organize as a result of MT-associated motor enzymes, the idea of MT initiation and/or stabilization in response to gradients that emanate from chromosomes, and the concept that a non/MT "matrix" guides the formation and organization of MTs. The data should eliminate or at least erode one or more of these models and will certainly provide baseline structural information for the interpretation of functional experiments on spindle formation and function that are being carried out in our labs and elsewhere.